Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

Introduction  

Although transforming growth factor β1 (TGFβ1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFβ1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFβ1 stimulation.     

Preparation of validoxylamine A by biotransformation of validamycin A using resting cells of a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

A gene encoding β-glucosidase was cloned and over-expressed in Escherichia coli. Validamycin A was then biotransformed into validoxylamine A by using the resting recombinant cells. The biotransformation yield reached 92% when the reaction was performed at 37°C for 1 h in the presence of 100 ml sodium phosphate buffer (0.1 M, pH 7.0), 32 mM validamycin A and 0.71 mg dry cell w/ml.     

Thrombin inhibitors with novel P1 binding pocket functionality: free energy of binding analysis

The high incidence of thrombembolic diseases justifies the development of new antithrombotics. The search for a direct inhibitor has resulted in the synthesis of a considerable number of low molecular weight molecules that inhibit human α-thrombin potently. However, efforts to develop an orally active drug remain in progress as the most active inhibitors with a highly basic P1 moiety exhibit an unsatisfactory bioavailability profile. In our previous work we solved several X-ray structures of human α-thrombin in complexes with (1) novel bicyclic arginine mimetics attached to the glycylproline amide and pyridinone acetamide scaffold and (2) inhibitors with a novel aza scaffold and with charged or neutral P1 moieties. In the present contribution, we correlate the structures of the complex between these inhibitors and the protein with the calculated free energy of binding. The energy of solvation was calculated using the Poisson–Boltzmann approach. In particular, the requirements for successful recognition of an inhibitor at the protein’s active site pocket S1 are discussed. Figure We report here on free energy of binding analysis of thrombin inhibitors with novel aza scaffold and novel bicyclic arginine mimetics in S1 pocket of thrombin     

Effect of <Emphasis Type="Italic">poxB</Emphasis> gene knockout on metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O₂ consumption and CO₂ evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.     

Isolation and properties of a <Emphasis Type="Italic">levo-</Emphasis>lactonase from <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> ECU2002: a robust biocatalyst for production of chiral lactones

A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration, acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50°C and 7.5, respectively, for γ-butyrolactone hydrolysis. The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which α-hydroxy-γ-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to γ-butyrolactone. The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including β-butyrolactone, α-hydroxy-γ-butyrolactone, α-hydroxy-β,β-dimethyl-γ-butyrolactone (pantolactone), and β-hydroxy-γ-butyrolactone, affording (+)-hydroxy acids in high (94.8∼98.2%) enantiomeric excesses (ee) and good conversions (38.2∼44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording the product in 94∼97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized.     

Unreported yet massive deforestation driving loss of endemic biodiversity in Indian Himalaya

Deforestation is a primary driver of biotic extinctions in the tropics. The impacts of deforestation in tropical biodiversity hotspots are of particular concern because these regions contain high concentrations of globally endemic species. However, the effects of large-scale deforestation on native biotas within the biodiversity hotspot of Himalaya remain poorly documented. Here we report on an alarming trend of deforestation in the Indian Himalaya and project the likely consequential extinctions of endemic taxa (species and subspecies) by 2100 across a broad range of taxonomic groups, including gymnosperms, angiosperms, fishes, amphibians, reptiles, birds, and mammals. With the current level of deforestation, by 2100 only about 10% of the land area of the Indian Himalaya will be covered by dense forest (>40% canopy cover)—a scenario in which almost a quarter of the endemic species could be wiped out, including 366 endemic vascular plant taxa and 35 endemic vertebrate taxa. We also show that inaccurate reporting of forest cover data by governmental institutions can result in underestimations of the biological impacts of deforestation, as well as potential miscalculations in land-use decisions (e.g., the construction of hydroelectric dams). Large-scale conservation efforts, including forest protection and reforestation, are urgently needed to avoid the impending deforestation-driven biodiversity losses in the Himalaya.     

Non-indigenous insect species in Israel and adjacent areas

Non-indigenous species cause great damage worldwide. Non-indigenous insects are known as harmful in many regions, but few comprehensive works have investigated non-indigenous insects as a group. We compiled a comprehensive database of established non-indigenous (ENI) insects in Israel and adjacent regions to investigate how they arrived, their biological characteristics, and the attributes of areas they invade. Of 218 species of ENI insects in this region, 124 are widespread. Most species came as stowaways, but 38 were brought intentionally for biological control. Most ENI insects in this region are in the Coleoptera, Diptera, Hymenoptera, Lepidoptera, and Homoptera. Species from various orders differ in their tendency to be localized or widespread, and in biogeographic origins. The distribution of species among orders differs between native and ENI insects. The Coastal Plain houses the most ENI insect species and the Negev and Judean deserts the fewest. Most ENI insects spread from the Coastal Plain to other regions. Absence of roads, settlements and presence of nature reserves are negatively correlated with occurrence of ENI species. Seventy-nine species are categorized as pests that damage produce, merchandise, forestry, etc. Despite a general dearth of knowledge on impacts of ENI insects on natural systems, 42 species are known to feed on native plants, some of conservation concern. Biological control agents are usually more limited in their distribution than other ENI insects. Further research, legislation, and enforcement are required to minimize effects of these species on agriculture and natural habitats.     

Environmental changes in Lake Taihu during the past century as recorded in sediment cores

The Lake Taihu drainage basin is an economically developed area with some of the highest population densities in China. The lake has deteriorated due to ecological destruction and eutrophication. Three short sediment cores from eastern, northeastern and southwestern Lake Taihu were collected. Total organic carbon (TOC), total nitrogen (TN), pigments, elements and particle size were analyzed for the purpose of understanding past trophic status and pollution levels. Sedimentation rates were based on ¹³⁷Cs or ²¹⁰Pb methods. Results indicated that sediment particle size became coarser since the 1920s, and the lake was contaminated by heavy metals, such as Cu and Zn, since the 1970s. A remarkable increase in eutrophication since the 1980s due to increased loading of untreated effluents from industry, agriculture and urbanization is reflected by total organic carbon, total nitrogen and pigments in the studied cores. However the onset times of eutrophication in different parts of Lake Taihu were not synchronous.     

A novel technique to study pore-forming peptides in a natural membrane

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na⁺:Ca²⁺,K⁺ exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K⁺, Na⁺ or Ca²⁺), the OS membrane resistance (R _m ) was typically larger than 1 GΩ in the presence of 1 mM external Ca²⁺. Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in ∼50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.     

Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload,Reactive Oxygen Species Production,Glutathione Depletion and Oxidative DNA Damage

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)₂, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)₂ or Cu²⁺ alone. It was found that the apoptosis induced by Cu(OP)₂ could not be induced by Cu²⁺ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)₂ in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)₂.